Role of CD5/CD5L interactions in the homeostasis of regulatory lymphocyte subpopulations and the control of autoimmune disorders

Resumen del trabajo presentado al: "6th European Workshop on Immune-Mediated Inflammatory Diseases" celebrado en Niza (Francia) del 23 al 25 de noviembre de 2011.-- et al.Peer Reviewe

Transgenic Expression of Soluble Human CD5 Enhances Experimentally-Induced Autoimmune and Anti-Tumoral Immune Responses

CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L) of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EμTg), expressing a circulating soluble form of human CD5 (shCD5) as a decoy to impair membrane-bound CD5 function. These shCD5EμTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE), as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma). This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+), and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EμTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens

Analysis of total cell numbers in primary and secondary lymphoid organs from shCD5EμTg mice.

<p>Total number of mononuclear cells in spleen, thymus, bone marrow, lymph node and peritoneal lavage samples was measured by flow cytometry using forward and side scatter for gating. ** p = 0.0018.</p

Exacerbated autoimmune disease in shCD5EμTg mice.

<p>A) Analysis of CIA induction in (DBA_B6)F1×shCD5EμTg mice and non-transgenic littermates. 7–8 week old mice were immunized s.c. near the base of the tail with 100 µl of antigen (2 mg/ml type II bovine collagen) emulsified in CFA plus Arthrogen-CIA adjuvant. Radiological signs were scored weekly for each mouse and results for each group averaged to determine overall clinical score. B) IL-6 levels were determined by real time RT-PCR in joint tissue, normalizing results to GAPDH expression levels. Results are expressed in arbitrary units (A.U.). * p = 0.05. C) and D) Antibodies against type II collagen were measured by ELISA in the sera of (DBA_B6)F1×shCD5EμTg transgenic mice and non-transgenic littermates. Antibody determination included total IgG (C) and IgG1 (D). Results are expressed as OD 450 nm. E) Analysis of EAE induction in shCD5EμTg and non-transgenic mice. 8–10 week old mice were administered s.c. with 200 µl of antigen (50 µg MOG peptide) in CFA. At the time of immunization and 48 hs later, 200 ng of Pertussis toxin were also injected i.p. Clinical score was determined daily and it represents the average value for each mice, where 0 = no disease; 0.5 = partially motionless tail; 1 = motionless tail; 2 = ataxia; 3 = loss of mobility in one limb; 4 = loss of mobility in back limbs; 5 = moribund. Graphs show the comparison between clinical scores of shCD5EμTg (n = 5) and non-transgenic mice (n = 10). p = 0.05 (Two-way ANOVA test). F) Demyelination and infiltration scores, as detected by solochrome cyanin and hematoxylin and eosin staining, respectively. ** p = 0.02.</p

Delayed tumour growth in shCD5EμTg mice.

<p>A) shCD5EμTg mice (n = 16) and non-transgenic littermates (n = 15) were injected s.c. with 5×10⁴ B16 melanoma cells. Tumour growth was recorded over time by measuring with a Vernier calliper. *** p = 0.0177. B) Experimental metastasis assay. Mice were injected i.v. with 1×10⁵ B16 melanoma cells. Fifteen days later, mice were sacrificed, their lungs removed and the metastases on the surface counted. p = 0.0994. C) Non-transgenic C57Bl/6 mice (n = 5 for each group) were injected with B16 cells as before. Chemotherapy (vincristine 0.5 mg/kg, Doxorubicin 3.3 mg/kg) was administered i.p. at day 3 after tumor cell injection. Additionally, rshCD5 (25 µg) or vehicle was injected i.p. every 48 hs for the duration of the experiment (20 days). ** p = 0.0083.</p

Flow cytometry analysis of B lymphocyte subpopulations in shCD5EμTg mice.

<p>A) To analyze peripheral B cell subsets from spleens of shCD5 transgenic mice and non-transgenic littermates, we used a gating strategy adapted from Cariappa <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084895#pone.0084895-Cariappa1" target="_blank">[45]</a>. Gated on IgM^highIgD^low, transitional 1 (T1), transitional 2 (T2) and marginal zone B cells can be distinguished based on their differential expression of the surface markers CD23 and CD93/AA4. *** p≤0.0003 * p = 0.0221. B) Cells obtained from peritoneal lavage samples were stained to distinguish B1a cells (B220+IgM^highIgD^lowCD5+), B1b cells (B220+IgM^highIgD^lowCD5−) and B2 cells (B220+IgM^highIgD^highCD5−) in shCD5 transgenic mice and non-transgenic littermates. * p = 0.0294 ** p = 0.0016.</p

Generation and characterization of human soluble hCD5 transgenic mice (shCD5EμTg).

<p><b>A</b>) Structure of the pIgHSV40shCD5 targeting vector. The cDNA sequence for shCD5 was inserted as <i>Xho</i>I-<i>Apa</i>I in between the promoter and polyA sequence of the pIg vector and then isolated as ScaI-ApaLI fragment for oocyte injections. <b>B</b>) Western blot analysis of human CD5 immunoprecipitates from transgenic (shCD5EμTg) and non-transgenic mice (NonTg) sera. The anti-CD5 Cris-1 mAb coupled with CNBr-activated Sepharose beads was used as immunoprecipitating agent. Western blotting was then carried out using the Leu-1 mAb plus HRP-labeled anti-mouse Ig. As a positive control, purified rshCD5 (50 ng) was included. <b>C</b>) ELISA detection of shCD5 in sera from shCD5EμTg mice. Sera from transgenic (shCD5EμTg, white) and non-transgenic littermates (NonTg, black) mice were analyzed by sandwich ELISA using Cris-1 and biotin-labeled Leu-1 as capture and developing mAbs, respectively. A standard rshCD5 curve was also analyzed in parallel to quantify results. <b>D–E</b>) shCD5EμTg mice are tolerant to exogenously administered rshCD5 but not rshCD6. shCD5EμTg mice and wild-type littermates were immunized twice with rshCD5 (<b>D</b>) or rshCD6 (<b>E</b>) (25 µg) in Freund's adjuvant (complete and incomplete, sequentially) within a 3-week interval. Mouse sera were collected 2 weeks after the boosting and added to ELISA plates coated with purified rshCD5, rshCD6 or BSA, then developed with a HRP-labeled anti-mouse IgG. Values represent the mean OD 450 nm values ± SD obtained in triplicate determinations for each sample (five mice per group). The total concentration of IgG was similar for both groups.</p